Programmed cell death has been shown to be an essential feature of negative selection of autoreactive lymphocytes and regulation of both physiological and pathological immune responses. Fas, a member of the TNF-receptor superfamily also known as CD95, has been shown to be important in apoptosis of activated T and B lymphocytes initiated by signaling through their antigen receptors. Humans and mice with germ line dominant-negative mutations in Fas accumulate abnormal lymphocytes and develop systemic autoimmunity similar to patients with Systemic Lupus Erythematosus. While most patients with non-familial autoimmune disease do not carry Fas mutations, there is evidence that Fas-mediated apoptosis may be impaired in the milieu of chronic inflammation. We have been investigating what signals regulate Fas-mediated apoptosis in T cells, with the eventual aim of harnessing these discoveries to modulate Fas-induced apoptosis for therapeutic goals in human disease. In activated CD4+ T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. We have identified the RhoGTPases Rac1 and Rac2 as essential components in RICD. RNAi-mediated knockdown of Rac GTPases greatly reduced Fas-dependent TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of ERM-family cytoskeletal linker proteins and translocation of Fas to lipid raft microdomains. In primary activated CD4+ T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases. We are now investigating heterogeneity in the ability of various T cell subsets to undergo Fas-mediated apoptosis and the molecular mechanisms underlying this heterogeneity. The goal in understanding these mechanisms is to design specific therapies to sensitize autoreactive lymphocytes to Fas-mediated apoptosis, which could constitute a long-acting and potentially permanent treatment for various autoimmune diseases such as Systemic Lupus, Multiple Sclerosis, Rheumatoid Arthritis, Type-I diabetes, and others in which autoreactive lymphocytes play a role. Through collaborations with investigators at NIH studying patients with the Autoimmune LymphoProliferative Syndrome (ALPS), a rare disorder associated with dominant-interfering Fas mutations, and the more common polygenic autoimmune disease Systemic Lupus Erythematosus (SLE), we are investigating translational implications of these findings.[unreadable] [unreadable] In another project related to biology the biology of Fas and Fas Ligand, we are investigating whether defects in the RICD pathway may be part of the predisposition to autoimmune disease development in the Wiskott-Aldrich syndrome, a genetic immunodeficiency with an unusually high incidence of autoimmune complications. Wiskott-Aldrich syndrome (WAS) is a X-linked disorder caused by mutations in the WAS gene and affecting the expression of the WAS protein (WASp) Despite concurrent immunodeficiency, autoimmunity occurs in 40-70% of WAS patients. We have found that WASp-deficient mice older than 6 months produce autoantibodies, and develop proliferative glomerulonephritis with immune complex deposition. Activated CD4+ T lymphocytes from WASp-deficient mice had reduced apoptosis after restimulation through the TCR, due to a specific defect in Fas Ligand (FasL) secretion. These results suggest a novel role of WASp in FasL secretion and argue that WASp-deficient mice provide a good model for the study of autoimmune manifestations of WAS and the development of immunological and gene therapies for these complications. [unreadable] [unreadable] In a collaboration with Raif Geha's laboratory at Childrens Hospital in Boston, we are investigating the mechanisms by which mutations in TACI, a TNF-family receptor important for regulating B cell survival and class-switching, cause familial cases of common variable immunodeficiency.